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Biochemical and molecular analyses of entry-point enzymes of phenylpropanoid metabolism in poplar (Populus trichocarpa x Populus deltoides) Ro, Dae-Kyun

Abstract

Cinnamate-4-hydroxylase (C4H) and its redox partner NADPHcytochrome P450 reductase (CPR), together with phenylalanine ammonia-lyase (PAL), play central roles at the gateway into plant phenylpropanoid metabolism. C4H catalyzes conversion of cinnamate to pcoumarate and has been proposed to anchor a multienzyme complex (MEC) on the endoplasmic reticulum (ER), recruiting PAL and potentially other soluble enzymes. The formation of p-coumarate is a key step in commitment of large amounts of carbon to lignin and soluble phenylpropanoid biosynthesis, especially in woody plants. In this thesis, catalytic and structural roles of poplar (Populus trichocarpa x P. deltoides) C4H and CPR were investigated. Clones of three isoforms of CPR were isolated from poplar xylem and young leaf cDNA libraries. Two of these cDNA clones and a previously cloned C4H cDNA were expressed in yeast. Efficient conversion of cinnamate to p-coumarate by yeast microsomes containing C4H confirmed the authenticity of the C4H cDNA, while co-expression of C4H and CPR substantiated the bona fide CPR activity of the two divergent CPRs. To examine whether the C4H and CPR are localized to the ER, C-terminal green fluorescent protein tagged versions of C4H and the two CPRs were expressed in Arabidopsis. Confocal microscopy analysis of Arabidopsis seedlings demonstrated predominant localization of the chimeric proteins on ER. To test the MEC model, an engineered yeast strain expressing PAL, C4H, and CPR was generated, in which phenylpropanoid product formation and metabolite channeling between enzymes could be investigated. Quantitative measurements showed that the triplegene expresser synthesized a striking amount of p-coumarate, while PAL-alone and C4Hinhibited triple expressers could not efficiently convert phenylalanine (Phe) to cinnamate. When 3H-Phe and 14C-cinnamate were simultaneously fed to the triple expresser, endogenously synthesized 3H-cinnamate was not preferred by C4H over 14C-cinnamate. Therefore, the observed efficient carbon flow from Phe to p-coumarate via the reaction catalyzed by PAL and C4H does not appear to require channeling through a MEC in yeast. Analysis of the biochemical properties of the entry point reactions and enzymes suggested instead that kinetic and thermodynamic coupling of PAL and C4H is sufficient to drive carbon-flux from primary metabolism to the phenylpropanoid pathway.

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